This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cellulose is a linear polymer of glucose and the most abundant biological material on Earth. As such, it is a vast, renewable resource for humanity's energy needs, both for fuels and power. In Nature, bacteria and fungi employ different strategies to convert recalcitrant cellulose into glucose. Many bacteria use large, extracellular enzyme complexes, cellulosomes, which consist of cellulose-degrading enzymes non-covalently bound to long peptide scaffolds. Fungi, conversely, use non-complexed enzyme cocktails to digest cellulose. Substantial US and international research efforts are focused on engineering enzymes from both the bacterial and fungal digestion strategies to convert cellulosic biomass into sugars, which can then be converted into renewable biofuels via fermentation or other routes. With significant focus on designing industrial biofuels processes, understanding of these two cellulose conversion strategies at the molecular level will enhance our ability to engineer enzymes for cost effective biofuels. Although structures of cellulase enzyme domains have been solved, there are relatively few studies that probe the solution structure of fungal enzymes or large, complexed cellulosomes. Thus, here we propose a 2-part study to examine the solution conformations of cellulose-degrading enzymes with SAXS. We will examine enzymes from the cellulosomal bacterium Clostridium thermocellum. The SAXS experiments for the cellulosomal systems include (1) a single enzyme complex with 6 domains, CbhA, that provides significant hydrolytic potential (Figure 2);(2) an isolated fibronectin domain from CbhA for which the function is unknown (Figure 3);and (3) scaffolds with binding sites populated with enzyme complexes such as Family 48/5/9 cellulases, the CbhA complex, and the scaffolds alone. Overall, this work will support our ongoing modeling efforts and drive rational protein engineering approaches to design enhanced cellulosomes for biofuels applications in the DOE BER-sponsored BioEnergy Science Center.